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Abstract:

This application relates to methods for ligating oligonucleotides having
complementarity to a target nucleic acid, and amplifying the ligated
oligonucleotides, where ligation and amplification occur in the same
reaction mixture.

Claims:

1. A method for ligating at least two oligonucleotides to produce a
ligated oligonucleotide and amplifying the ligated oligonucleotide,
wherein ligation and amplification occur in a single reaction mixture.

2. The method of claim 1 whereby a ligand is detected in a test sample,
the method comprising the steps of, in combination: a) contacting a
target protein or analyte with at least a first and second probe, each
probe having binding specificity for the protein or analyte, and being
adjoined to at least one type of oligonucleotide; b) ligating the
oligonucleotides on the first and second probes to one another using a
ligase to produce a target nucleic acid and amplifying the target nucleic
acid; and, c) detecting the amplified target nucleic acid.

3. The method of claim 1 or 2, wherein a portion of at least one of said
probes is an antibody.

4. The method of claim 3, wherein a portion of each of said first and
second probes are antibodies.

5. The method of any one of claims 1-4, wherein at least one of said
oligonucleotides comprises at least three nucleotides.

6. The method of any one of claims 1-5, wherein at least one of said
oligonucleotides consists of three nucleotides.

7. The method of any one of claims 1-6, wherein said oligonucleotides are
ligated using a small footprint ligase (SFL).

8. The method of claim 7, wherein said small footprint ligase is
contacted with adenosine triphosphate prior to use.

9. The method of any one of claims 1-8, wherein said ligated
oligonucleotide is amplified using the polymerase chain reaction (PCR).

14. A method for detecting a target in a sample wherein: a) binding a
first and a second probe, each of which binds specifically to the target,
wherein each of the probes comprises an oligonucleotide portion or tail;
b) ligating the first and second oligonucleotide tails thereby producing
a ligated oligonucleotide template; and, c) performing a polymerase chain
reaction (PCR) of the oligonucleotide template across the first and
second oligonucleotide to quantify the said template.

15. The method of claim 14, wherein steps b and c are performed in the
same reaction mixture.

16. The method of claim 14 or 15, wherein a splint oligonucleotide is
used to ligate said first and second oligonucleotide tails.

17. The method of claim 16, wherein the 3' and 5' ends (which overlap
with said first and second oligonucleotide tails, respectively) of said
splint oligonucleotide are symmetrical or asymmetrical to one another.

18. The method of claim 16 or 17, wherein said splint oligonucleotide is
blocked at it's 3'-end.

19. The method of any of claims 16-18, wherein said splint
oligonucleotide is at least 6 nucleotides in length.

20. The method of any of claims 16-19, wherein said 3'-end of said splint
oligonucleotide overlaps with at least 3 nucleotides of said first
oligonucleotide tail and/or said 5'-end of said splint oligonucleotide
overlaps with at least 3 nucleotides of said second oligonucleotide tail.

21. The method of any of claims 14-20, wherein the length of said first
and second oligonucleotide tails is at least 3 nucleotides in length.

22. The method of any of claims 14-21, wherein a small footprint ligase
(SFL) is used to ligate said first and second oligonucleotide tails.

23. The method of any of claims 14-22, wherein said first and/or second
probes further comprises an antibody portion specific to said target.

24. A method for detecting a target in sample comprising: a) binding a
first and a second probe, wherein each probe binds specifically to the
target, each of said probes comprise an oligonucleotide portion or tail;
b) ligating the oligonucleotides to produce a ligated oligonucleotide
template and amplifying the template by PCR in a single step to produce
an amplified template; and, c) quantitating the amplified template

24. The method of claim 24, wherein the probe further comprises an
antibody.

27. The method of claim 26, wherein the ligase is a small-footprint DNA
ligase (SFL).

28. The method of any one of claims 24-27, wherein said oligonucleotides
are ligated using a splint oligonucleotide.

29. The method of claim 28, wherein said splint oligonucleotide comprises
a 3' end of four to nine bases in length and a 5' end of four to nine
bases in length.

30. The method of claim 28 or 29, wherein said 3' and 5' ends (which
overlap with said first and second tails, respectively) of said splint
oligonucleotide are symmetrical or asymmetrical to one another.

31. The method of claim 28-30, wherein said splint oligonucleotide is
blocked at it's 3'-end.

32. The method of any one of claims 23-31, wherein the ligase is
pre-enriched using ATP.

33. The method of any one of claims 23-32, wherein said ligase is
inactivated after ligation using a protease or heat.

34. The method of any one of claims 23-33, wherein said template is
quantified using real-time PCR.

35. The method of claim 34, wherein said real-time PCR assay is a TaqMan
assay.

36. The method of claim 1, 14, or 24, wherein said method provides at
least about a one to three-fold dCt improvement over a typical PLA method
or process.

37. The method of claim 1, 14, or 24, wherein said method provides about
a two- to ten-fold improvement/increase in sensitivity over the typical
process.

38. The method of claim 36 or 37, wherein said typical method or process
includes the use of a protease and/or dilution of the reaction mixture
prior to PCR and said improved method of claim 1, 14, or 24 does not.

39. The method of claim 2, wherein said oligonucleotides on said first
and second probes, are at least partially complementary to one another

Description:

FIELD OF THE DISCLOSURE

[0001] This application relates to methods for ligating oligonucleotides
having complementarity to a target nucleic acid, and amplifying the
ligated oligonucleotides, where ligation and amplification occur in the
same reaction mixture.

BACKGROUND OF THE DISCLOSURE

[0002] The correlation of gene and protein expression changes in
biological systems has been hampered by the need for separate sample
handling and analysis platforms for nucleic acids and proteins. In
contrast to the simple, rapid, and flexible workflow of quantitative PCR
(qPCR) methods, which enable characterization of several classes of
nucleic acid biomarkers (e.g., DNA, mRNA, and microRNAs), protein
analysis methods such as Western blotting are cumbersome, laborious, and
much less quantitative. Proximity Ligation Assays (PLAs) have been shown
to eliminate some of these problems. However, improvements to PLAs are
desired by those of skill in the art.

[0003] Typical or conventional PLAs usually involve at least three or four
steps. The first step is typically the binding of first and second probes
(e.g., antibody probes) to a ligand (e.g., a protein of interest) such
that the probes are in close proximity to another. Each of the probes
typically contain an oligonucleotide. The oligonucleotides are brought
into proximity to one another with the binding of the probes and, in the
second step, are then ligated to one another (e.g., the ligation event).
The ligated oligonucleotides may then be amplified and detected to
determine the presence of the ligand with a test sample (e.g., a
biological sample). This step is typically accomplished by adding
ligation components, such as ligase, adenosine triphosphate (ATP) and
buffer-salt mixture, to the binding reaction. In the third step, the
ligase is typically then deactivated (e.g., by protease digestion) to
prevent any further ligation of unbound oligonucleotides. In the fourth
step, the reaction mixture is transferred to a real-time polymerase chain
reaction (PCR) mixture and the quantity of the amplified product
determined by quantitative PCR (qPCR). As described below, it has been
surprisingly found that the third step (ligase digestion) may be
eliminated, thereby allowing ligation and amplification to occur in the
same reaction mixture without inactivation of the ligase. These and other
features and advantages of the methods described herein will be apparent
to the skilled artisan from this disclosure.

SUMMARY OF THE DISCLOSURE

[0004] Provided herein are methods for ligating and amplifying
oligonucleotides. In some embodiments, the oligonucleotides are attached
to ligand-specific probes, and amplification of the oligonucleotides
indicates that the probes have bound a ligand in the sample. In one
embodiment, a method for ligating at least two oligonucleotides to
produce a ligated oligonucleotide and amplifying the ligated
oligonucleotide, wherein ligation and amplification occur in a single
reaction mixture (e.g., that may be considered undiluted) is provided. In
some embodiments, a third oligonucleotide may be used to bridge the at
least two oligonucleotides that are bound to the probes. In certain
embodiments, the method may comprise detecting a ligand in a test sample
(e.g., a biological sample) comprising contacting the protein with at
least a first and second probe, each probe having binding specificity for
the protein and being adjoined to at least one type of oligonucleotide,
the oligonucleotides on the first and second probes, respectively, being
at least partially complementary to one another; ligating the
oligonucleotides on the first and second probes to one another using a
ligase to produce a target nucleic acid and amplifying the target nucleic
acid; and, detecting the amplified target nucleic acid. For instance, the
method may comprise detecting a protein in a test sample, the method
comprising contacting the protein with at least two probes having binding
specificity therewith, each of the two agents comprising at least one
oligonucleotide; ligating the oligonucleotides to produce a ligated
oligonucleotide and amplifying the ligated oligonucleotide in a single
reaction mixture; and, detecting amplification of the ligated
oligonucleotide. In some embodiments, one or more of the probes is an
antibody. In certain embodiments, at least one of the oligonucleotides
comprises at least three nucleotides. Some embodiments provide for the
oligonucleotide being ligated using a small footprint ligase, which may
be contacted with adenosine triphosphate prior to use. Any type of
amplification procedure may be used such as, without limitation,
polymerase chain reaction (PCR) (e.g., quantitative PCR (qPCR)). In some
embodiments, it may be beneficial to inactivate the ligase prior to
amplification (e.g., using a protease). Other embodiments of the methods
described herein will be apparent to the skilled artisan from the
disclosure provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The skilled artisan will understand that the drawings described
below are for illustration purposes only. The drawings are not intended
to limit the scope of the present teachings in any way.

[0011] FIG. 6. Use of the improved PLA process with various target nucleic
acids.

[0012] FIG. 7. Comparison of two different splint lengths at varying
concentrations.

[0013] FIG. 8. Comparison of five different splint lengths.

[0014] FIG. 9. Comparison of T4 ligase to two different SF ligases (e.g.,
SF and DLxD).

DETAILED DESCRIPTION

[0015] Disclosed herein are methods for performing proximity ligation
assays. In typical or conventional proximity ligation assay (PLA)
processes (FIG. 1), a probe mix and sample are combined into a binding
reaction. Following the binding reaction, the ligation reaction mixture
is added in order to carry out the ligation reaction. To prepare the
ligation reaction mixture, a ligase and ligation buffer are diluted.
Following the ligation reaction, the ligated product is stabilized by
protease digestion; the protease is then inactivated (e.g, using heat). A
portion of the ligated product is transferred to a real-time PCR reaction
mixture, then placed on a PCR reaction vessel (e.g., plate) in a qPCR
instrument. Detection and quantification of the ligated product then
proceeds using standard techniques.

[0016] In one embodiment of the improved PLA process disclosed herein, a
cell lysate may be prepared and a ligation buffer added thereto. To that
mixture may then be added a proximity probe mixture, a ligase, and a PCR
mixture (which may include, for example, a thermostable polymerase). This
combined reaction mixture can then incubated for a suitable amount of
time (e.g., one hour, 37° C.), the ligase optionally inactivated
(e.g., using heat) and PCR performed directly on the mixture. A schematic
of an exemplary embodiment of the improved PLA process is illustrated in
FIG. 2. As shown therein, the binding reaction is the same as that shown
in FIG. 1. However, in some embodiments of the improved PLA processes,
the ligase is added to the real-time PCR mixture which is then added
directly to the binding reaction. In some embodiments, this reaction
mixture is then deposited onto a reaction plate and then analyzed by a
qPCR instrument. Detection and quantification of the ligated product then
proceeds using standard techniques.

[0017] In some embodiments, the lesser dilution factor provided thereby
may result in a higher PLA probe concentration in the ligation reaction.
The increased probe concentration may cause increased background signal,
which may be minimized by using a short splint oligonucleotide at reduced
concentration. For instance, a suitable splint oligonucleotide may be 14
nucleotides in length (e.g., at least five nucleotides in the 3' end and
at least nine nucleotides in the 5' end; 5+9). To ensure the ligation
efficiency, a small footprint ligase (SFL) may be used. In some
embodiments, to further simplify the ligation-PCR step, the typical
addition of ATP to the ligation reaction may be omitted. Instead, one may
optionally use an ATP-enriched SFL (e.g., an SFL that is exposed to or
contacted with an abundance or additional supply of ATP for some period
of time). This enrichment step may be especially useful when
co-substrates for other ligases are used. Thus, the binding reaction may
be assembled by combining proximity probes and samples containing target
molecule(s), and incubating the mixture such that binding between the
probes and the target molecule(s) occurs. In some embodiments, after the
binding reaction, a ligation-PCR mix (e.g., comprising a short splint
oligo (e.g., an oligonucleotide that is at least 6 nucleotides, e.g., 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides)
in length, an SFL, and standard real-time PCR components) may be added.
In some other embodiments, the ligation reaction may then take place at
room-temperature, and the product amplified and quantitated by real-time
PCR. In some embodiments, the ligase may be deactivated (e.g., using
heat). The ligated product may then be subjected to real-time PCR
immediately or following storage. Thus, the various embodiments of the
novel work flows for improved PLA processes as disclosed herein provide
reduced dilution factors which enable one to accomplish ligation and PCR
in a single reaction mixture (e.g., or in a single step without
intermediate/intervening steps). In some preferred embodiments, a short
splint oligonucleotide and an SFL may also be used to control any
increased background reactions. In additional embodiments, the SFL may be
pre-enriched using ATP.

[0018] Exemplary typical and improved PLA processes are further compared
in FIGS. 3A and 3B. As shown therein, the typical processes include
sample preparation, a binding reaction, ligation, ligase inactivation
using a protease, protease inactivation (e.g., using heat), followed by
real-time PCR. To carry out the PCR step in typical PLA processes, a
portion of the reaction mixture containing the inactivated ligase and
protease is usually transferred to a PCR plate, and the "PCR mix" (e.g.,
containing primers, dNTPs, polymerase, and the like) added thereto. As
shown in FIG. 3B, the disclosed improved processes can eliminate the use
of a protease and dilution of the reaction mixture prior to PCR. As shown
therein, the ligase may be inactivated using, for example, heat, and the
resultant reaction mixture placed directly into a qPCR assay/instrument.
Thus, simplified, improved PLA work flows can use entire binding reaction
products in a real-time PCR assay (e.g., in a multi-plate well). This
provides an improved work-flow and reduced dilution of the reaction
mixture. As a result, in some embodiments of the improved PLA processes,
the PCR reaction mixture contains a higher concentration of the ligated
product (e.g., the target nucleic acid). In some embodiments, the
improvement provided by the improved PLA processes can be measured as the
dCT of the reaction (e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
signal (dCT); see, for example, FIG. 4). Sensitivity of the improved PLA
processes as compared to typical PLA processes can also be observed
(e.g., as fold-change; see, for example, FIG. 5).

[0019] The processes described herein provide for, in some embodiments,
detecting a protein in a test sample, the methods comprising contacting
the protein with at least two probes having binding specificity
therewith, each of the two probes comprising at least one
oligonucleotide; ligating the oligonucleotides to produce a ligated
oligonucleotide; amplifying the ligated oligonucleotide in a single
reaction mixture; and, detecting amplification of the ligated
oligonucleotide. In some embodiments, a third oligonucleotide may also be
used to bridge each of the oligonucleotides attached to each of the
probes. In some embodiments, one or more of the probes is an antibody. In
certain embodiments, at least one of the oligonucleotides comprises at
least three nucleotides. Some embodiments provide for use of a SFL, which
may optionally be contacted with adenosine triphosphate (ATP) prior to
use, for ligation of the oligonucleotides. Any type of amplification
procedure may be used such as, without limitation, polymerase chain
reaction (PCR) (e.g., quantitative PCR). In some embodiments, it may be
beneficial to inactivate the ligase prior to amplification (e.g., using a
protease, heat, or any other methods known in the art). Other embodiments
of the inventions described herein will be apparent to the skilled
artisan from the disclosure provided herein.

[0020] In some embodiments, a method for detecting a target in a sample is
provided where the method includes the steps of binding a first and a
second probe, each of which binds specifically to the target, wherein
each of the probes comprises an oligonucleotide portion (or tail);
ligating the first and second oligonucleotide tails thereby producing a
ligated oligonucleotide template; and, performing a polymerase chain
reaction (PCR) of the oligonucleotide template across the first and
second oligonucleotide tails to quantify the said template. In some
embodiments, the ligation and PCR steps may be performed in the same
reaction mixture. In other embodiments, the method may include binding a
first and a second probe, wherein each probe binds specifically to the
target, each of the probes comprise a oligonucleotide tail; ligating the
oligonucleotide tails to produce a ligated oligonucleotide template and
amplifying the template by PCR in a single step to produce an amplified
template; and, quantitating the amplified template. The probes may
comprise antibodies which specifically bind to the target. The
oligonucleotides may be ligated using a splint oligonucleotide (e.g.,
splint oligos of at least 6 nucleotides in length having 3' and 5'
overhangs of, for example, 9+9, 9+8, 9+7, 9+6, 9+5, 8+8, 5+3, 4+7, 3+3
nucleotides, or any other possible variations in length or symmetry as
contemplated and described in further detail below). In some embodiments,
the ligase may be pre-enriched using ATP and/or inactivated using, for
example, one or more proteases and/or heat. The amplified template may be
quantified by any suitable method including, for example, real-time PCR
(e.g., a TaqMan assay or a molecular beacon assay).

[0021] The improved processes disclosed and/or exemplified herein provide
reduced work times from process start time to collection of results
(e.g., faster), reduced hands-on time (e.g., simpler and cheaper),
reduced lab plasticware usage (e.g., cheaper and more environmentally
sound ("greener")), and increased signals and sensitivities (e.g., more
sensitive). In some embodiments, these improved processes provide
simplified work flows by combining ligation and PCR steps, reduced
dilution factors from the binding step to the ligation step, reduced
binding probe concentrations to enable reduced dilution factors, use of
shorter connector oligonucleotides (e.g., as few as 6 nucleotides in
length) to control background signals, use of lower connector
oligonucleotide concentrations to control background signals, use of SF
ligases to enable use of shorter connector oligonucleotide lengths,
ATP-enriched SF ligase purification schemes to omit ATP in ligation-PCR
step, and/or enabling use of the entire reaction volume to improve PLA
signal and sensitivity.

[0022] The methods described herein are particularly useful in that the
same may be used with various systems for detecting proteins. Exemplary
of such systems include, for example, TaqMan® Protein Assays.
TaqMan® Protein Assays are an adapted form of PLA®, a proximity
ligation assay technology that combines antibody-protein binding with
detection of the reporter nucleic acid by real-time PCR. Applied
Biosystems has optimized this technique for use with crude cell and
tissue lysates and combined it with TaqMan® chemistry to create a
highly sensitive and specific process for measuring protein expression in
small samples. Assays have been developed for the detection of OCT3/4,
NANOG, SOX2, and LIN28 in human embryonic stem cells, as well as ICAM1
and CSTB to measure relative quantification in human cells. The basic
steps of such assays include binding of a protein target by paired assay
probes, ligation of the oligonucleotides by a DNA ligase, and
amplification of the ligation product by TaqMan real-time PCR assay. The
probes used in the first step are typically target-specific antibodies
conjugated to oligonucleotides through a biotin-streptavidin (SA)
linkage. Each oligonucleotide in the pair presents a 5' or 3' end that
are brought into proximity when the assay probes bind to two different
epitopes on the target protein. The substrate for the ligase is typically
a bridge structure formed by hybridization of a third oligonucleotide to
the oligonucleotide ends of the assay probe pair. This structure forms
preferentially when the assay probes are in proximity to each other. The
ligation product typically serves the template in the TaqMan®
real-time PCR assay. The systems may be used to, for example, to perform
protein analysis on small samples (e.g., stem cells, germ cell tumors),
correlate and/or validate results from RNA and protein quantitation,
analyze post-translational modifications, validate siRNA-induced gene
silencing, and/or validate gene transfection/transduction experiments.
Data generated using these systems may be analyzed using software such
as, for instance, ProteinAssist® software package (Applied
Biosystems®).

[0023] To more clearly and concisely describe and point out the subject
matter of the present disclosure, the following definitions are provided
for specific terms, which are used in the following description and the
appended claims. Throughout the specification, exemplification of
specific terms should be considered as non-limiting examples.

[0024] As used herein the terms "nucleotide" or "nucleotide base" refer to
a nucleoside phosphate. It includes, but is not limited to, a natural
nucleotide, a synthetic nucleotide, a modified nucleotide, or a surrogate
replacement moiety or universal nucleotide (e.g., inosine). The
nucleoside phosphate may be a nucleoside monophosphate, a nucleoside
diphosphate or a nucleoside triphosphate. A "nucleotide" refers to a
nucleotide, nucleoside or analog thereof. Optionally, the nucleotide is
an N- or C-glycoside of a purine or pyrimidine base. (e.g.,
deoxyribonucleoside containing 2-deoxy-D-ribose or ribonucleoside
containing D-ribose). Examples of other analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides. Nucleotide bases
usually have a substituted or unsubstituted parent aromatic ring or
rings. In certain embodiments, the aromatic ring or rings contain at
least one nitrogen atom. In certain embodiments, the nucleotide base is
capable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an
appropriately complementary nucleotide base. Exemplary nucleotide bases
and analogs thereof include, but are not limited to, purines such as
2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine,
N6-Δ2-isopentenyladenine (61A),
N6-Δ2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine,
guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7
mG), 2-thiopyrimidine, 6-thioguanine (6sG) hypoxanthine and
O6-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and
7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C),
5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT),
5,6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and
5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and
4-methylindole; pyrroles such as nitropyrrole; nebularine; base (Y); etc.
In certain embodiments, nucleotide bases are universal nucleotide bases.
Additional exemplary nucleotide bases can be found, e.g., in Fasman,
1989, Practical Handbook of Biochemistry and Molecular Biology, pp.
385-394, CRC Press, Boca Raton, Fla., and the references cited therein. A
"universal base", as used herein, is a base that is complementary to more
than one other bases. Fully universal bases can pair with any of the
bases typically found in naturally occurring nucleic acids. The base need
not be equally capable of pairing with each of the naturally occurring
bases. Alternatively, the universal base may pair only or selectively
with two or more bases but not all bases. Optionally the universal base
pairs only or selectively with purines, or alternatively with
pyrimidines. If so desired, two or more universal bases can be included
at a particular position in a probe. A number of universal bases are
known in the art including, but not limited to, hypoxanthine,
3-nitropyrrole, 4-nitroindole, 5-nitroindole, 4-nitrobenzimidazole,
5-nitroindazole, 8-aza-7-deazaadenine,
6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P. Kong Thoo Lin. and
D. M. Brown, Nucleic Acids Res., 1989, 17, 10373-10383),
2-amino-6-methoxyaminopurine (D. M. Brown and P. Kong Thoo Lin,
Carbohydrate Research, 1991, 216, 129-139), etc. Hypoxanthine is one
preferred fully universal base. Nucleosides comprising hypoxanthine
include, but are not limited to, inosine, isoinosine, 2'-deoxyinosine,
and 7-deaza-2'-deoxyinosine, 2-aza-2'deoxyinosine. Naturally occurring
and synthetic analogs may also be used, including for example
hypoxanthine, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-N4
ethencytosine, 4-aminopyrrazolo[3,4-d]pyrimidine and
6-amino-4-hydroxy[3,4-d]pyrimidine, among others. The nucleotide units of
the oligonucleotides may also have a cross-linking function (e.g. an
alkylating agent).

[0025] A nucleoside is usually a compound having a nucleotide base
covalently linked to the C-1' carbon of a pentose sugar. In certain
embodiments, the linkage is via a heteroaromatic ring nitrogen. Typical
pentose sugars include, but are not limited to, those pentoses in which
one or more of the carbon atoms are each independently substituted with
one or more of the same or different --R, --OR, --NRR or halogen groups,
where each R is independently hydrogen, (C1-C6) alkyl or
(C5-C14) aryl. The pentose sugar may be saturated or
unsaturated. Exemplary pentose sugars and analogs thereof include, but
are not limited to, ribose, 2'-deoxyribose,
2'-(C1-C6)alkoxyribose, 2'-(C5-C14)aryloxyribose,
2',3'-dideoxyribose, 2',3'-didehydroribose, 2'-deoxy-3'-haloribose,
2'-deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose,
2'-deoxy-3'-aminoribose, 2'-deoxy-3'-(C1-C6)alkylribose,
2'-deoxy-3'-(C1-C6)alkoxyribose and
2'-deoxy-3'-(C5-C14)aryloxyribose. One or more of the pentose
carbons of a nucleoside may be substituted with a phosphate ester, as
disclosed in, for example, U.S. Pat. No. 7,255,994. In certain
embodiments, the nucleosides are those in which the nucleotide base is a
purine, a 7-deazapurine, a pyrimidine, a universal nucleotide base, a
specific nucleotide base, or an analog thereof. Nucleotide analogs
include derivatives in which the pentose sugar and/or the nucleotide base
and/or one or more of the phosphate esters of a nucleoside may be
replaced with its respective analog. Exemplary pentose sugar analogs and
nucleotide base analog are described above. Exemplary phosphate ester
analogs include, but are not limited to, alkylphosphonates,
methylphosphonates, phosphoramidates, phosphotriesters,
phosphorothioates, phosphorodithioates, phosphoroselenoates,
phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates,
phosphoroamidates, boronophosphates, etc., and may include associated
counterions. Other nucleotide analogs are nucleotide analog monomers
which can be polymerized into polynucleotide analogs in which the DNA/RNA
phosphate ester and/or sugar phosphate ester backbone is replaced with a
different type of linkage. Exemplary polynucleotide analogs include, but
are not limited to, peptide nucleic acids, in which the sugar phosphate
backbone of the polynucleotide is replaced by a peptide backbone. The
internucleoside linkages can be a phosphodiester linkage, although other
linkages (e.g., scissile linkages which can be substantially cleaved
under conditions in which phosphodiester linkages are not substantially
cleaved) can be used. For example, a linkage that contains an AP
endonuclease sensitive site, for example an abasic residue, a residue
containing a damaged base that is a substrate for removal by a DNA
glycosylase, or another residue or linkage that is a substrate for
cleavage by an AP endonuclease, or a disaccharide nucleoside.

[0026] As used herein, the term "oligonucleotide" ("oligo") or
"polynucleotide" may refer to an oligomer of nucleotides or derivatives
thereof. Polynucleotides include double- and single-stranded DNA, as well
as double- and single-stranded RNA, DNA:RNA hybrids, peptide-nucleic
acids (PNAs) and hybrids between PNAs and DNA or RNA, and also include
known types of modifications, for example, labels which are known in the
art, methylation, "caps," substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications such
as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoramidates, carbonates, etc.), with
negatively charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), and with positively charged linkages (e.g.,
aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing
pendant moieties, such as, for example, proteins (including nucleases,
toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with
intercalators (e.g., acridine, psoralen, etc.), those containing
chelators (e.g., metals, radioactive metals, boron, oxidative metals,
etc.), those containing alkylators, those with modified linkages (e.g.,
alpha anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide or oligonucleotide. The oligomers may also include
modified bases, and/or backbones (e.g., modified phosphate linkage or
modified sugar moiety). Non-limiting examples of synthetic backbones that
confer stability and/or other advantages to the oligomers may include
phosphorothioate linkages, peptide nucleic acid, locked nucleic acid
(Singh, et al. Chem Commum 4:455-456 (1998)), xylose nucleic acid, and/or
analogues thereof. In other cases, the polynucleotide can contain
non-nucleotidic backbones, for example, polyamide (e.g., peptide nucleic
acids (PNAs)) and polymorpholino (commercially available from the
Anti-Virals, Inc., Corvallis, Oreg., as Neugene® polymers), and other
synthetic sequence-specific nucleic acid polymers providing that the
polymers contain nucleobases in a configuration which allows for base
pairing and base stacking, such as is found in DNA and RNA.

[0027] Oligonucleotides and/or polynucleotides may be any length "n." For
example, n may be any of 1, 2, 4, 6, 8, 12, 16, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40 etc. number of nucleotides. The polynucleotide
structure (N)n represents an oligonucleotide consisting of n number
of nucleotides N (e.g., (I)8 is representative of an oligonucleotide
having the sequence IIIIIIII; or (A)12 is representative of an
oligonucleotide having the sequence AAAAAAAAAAAA). Other types of
oligonucleotides or polynucleotides may also be suitable for use as would
be understood to one of skill in the art from this disclosure.

[0028] Oligonucleotides and/or polynucleotides may optionally be attached
to one or more non-nucleotide moieties such as labels and other small
molecules, large molecules such proteins, lipids, sugars, and solid or
semi-solid supports, for example through either the 5' or 3' end. Labels
include any moiety that is detectable using a detection method of choice,
and thus renders the attached nucleotide or polynucleotide similarly
detectable using a detection method of choice (e.g., using a SGC and/or
detectable label). Optionally, the label emits electromagnetic radiation
that is optically detectable or visible. In some cases, the nucleotide or
polynucleotide is not attached to a label, and the presence of the
nucleotide or polynucleotide is directly detected.

[0029] As used herein, the term "nucleic acid" refers to polymers of
nucleotides or derivatives thereof. As used herein, the term "target
nucleic acid" refers to a nucleic acid that is desired to be amplified in
a nucleic acid amplification reaction. For example, the target nucleic
acid comprises a nucleic acid template. In some embodiments, the target
nucleic acid may be the product of the ligation of at least two
oligonucleotides to one another.

[0030] As used herein, the term "sequence" refers to a nucleotide sequence
of an oligonucleotide or a nucleic acid. Throughout the specification,
whenever an oligonucleotide/nucleic acid is represented by a sequence of
letters, the nucleotides are in 5' to 3' order from left to right. For
example, if the polynucleotide contains bases Adenine, Guanine, Cytosine,
Thymine, or Uracil, the polynucleotide sequence can be represented by a
corresponding succession of letters A, G, C, T, or U), e.g., a DNA or RNA
molecule. And, an oligonucleotide represented by a sequence
(I)n(A)n wherein n=1, 2, 3, 4 and so on, represents an
oligonucleotide where the 5' terminal nucleotide(s) is inosine and the 3'
terminal nucleotide(s) is adenosine.

[0031] Oligonucleotides and/or polynucleotides can optionally be regarded
as having "complementary" sequences if the same may hybridize to one
another. The term "hybridization" typically refers to the process by
which oligonucleotides and/or polynucleotides become hybridized to each
other. The adjectival term "hybridized" refers to two polynucleotides
which are bonded to each other by two or more sequentially adjacent base
pairings. Typically, these terms refer to "specific hybridization". Two
oligonucleotides and/or polynucleotides may selectively (or specifically)
hybridize to each other if they bind significantly or detectably to each
other under stringent hybridization conditions when present in a complex
polynucleotide mixture such as total cellular or library DNA. In some
embodiments, for selective or specific hybridization, a positive signal
is at least two times background, preferably 10 times background
hybridization. Optionally, stringent conditions are selected to be about
5-10° C. lower than the thermal melting point for the specific
sequence at a defined ionic strength pH. Stringent conditions are
optionally in which the salt concentration is less than about 1.0 M
sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or
other salts) at pH 7.0 to 8.3 and the temperature is at least about
30° C. for short probes (e.g., 10 to 50 nucleotides) and at least
about 60° C. for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5×SSC, and 1% SDS,
incubating at 42° C., or, 5×SSC, 1% SDS, incubating at
65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
Nucleic acids that do not hybridize to each other under stringent
conditions are still substantially identical if the polypeptides which
they encode are substantially identical. "Nonspecific hybridization" is
used to refer to any unintended or insignificant hybridization, for
example hybridization to an unintended polynucleotide sequence other than
the intended target polynucleotide sequence. The unintended
polynucleotide sequence can be on the same or different polynucleotide
from the intended target. In some cases, the only intended hybridization
can be from Watson-Crick base pairing between two polynucleotides. Other
kinds of intended base pairings can include base pairing between
corresponding analogs of such nucleotides or between iso-cytidine and
iso-guanine. In some cases where hybridization is only intended between
complementary bases, any bonding between non-complementary bases is
considered to be non-specific hybridization.

[0032] In some embodiments, complementary sequences may be those that,
when hybridized together, may be efficiently ligated to a third
polynucleotide that has hybridized adjacently to it. Similarly,
nucleotide residues can be regarded as complementary if when both are
base-paired with each other within two hybridized polynucleotides, either
nucleotide can be ligated in a template-driven ligation reaction when
situated as the terminal nucleotide in its polynucleotide. Nucleotides
that are efficiently incorporated by DNA polymerases opposite each other
during DNA replication under physiological conditions are also considered
complementary. In an embodiment, complementary nucleotides can form base
pairs with each other, such as the A-T/U and G-C base pairs formed
through specific Watson-Crick type hydrogen bonding between the
nucleobases of nucleotides and/or polynucleotides positions antiparallel
to each other. The complementarity of other artificial base pairs can be
based on other types of hydrogen bonding and/or hydrophobicity of bases
and/or shape complementarity between bases. In appropriate instances,
polynucleotides can be regarded as complementary when the same may
undergo cumulative base pairing at two or more individual corresponding
positions in antiparallel orientation, as in a hybridized duplex.
Optionally there can be "complete" or "total" complementarity between a
first and second polynucleotide sequence where each nucleotide in the
first polynucleotide sequence can undergo a stabilizing base pairing
interaction with a nucleotide in the corresponding antiparallel position
on the second polynucleotide. "Partial" complementarity describes
polynucleotide sequences in which at least 20%, but less than 100%, of
the residues of one polynucleotide are complementary to residues in the
other polynucleotide. A "mismatch" is present at any position in the two
opposed nucleotides that are not complementary. In some ligation assays,
a polynucleotide can undergo substantial template-dependent ligation even
when it has one or more mismatches to its hybridized template.
Optionally, the polynucleotide has no more than 4, 3, or 2 mismatches,
e.g., 0 or 1 mismatch, with its template. In some assays, the
polynucleotide will not undergo substantial template-dependent ligation
unless it is at least 60% complementary, e.g., at least about 70%, 80%,
85%, 90%, 95% or 100% complementary to its template.

[0033] "Degenerate", with respect to a position in a polynucleotide that
is one of a population of polynucleotides, means that the identity of the
base of the nucleoside occupying that position varies among different
members of the population. A population of polynucleotides in this
context is optionally a mixture of polynucleotides within a single
continuous phase (e.g., a fluid). The "position" can be designated by a
numerical value assigned to one or more nucleotides in a polynucleotide,
generally with respect to the 5' or 3' end. For example, the terminal
nucleotide at the 3' end of an extension probe may be assigned position
1. Thus in a pool of extension probes of structure 3'-XXXNXXXX-5', the N
is at position 4. A position is said to be k-fold degenerate if it can be
occupied by nucleosides having any of k different identities. For
example, a position that can be occupied by nucleosides comprising either
of 2 different bases is 2-fold degenerate.

[0034] A "solid support", as used herein, typically refers to a structure
or matrix on or in which ligation and/or amplification reagents (e.g.,
nucleic acid molecules, microparticles, and/or the like) may be
immobilized so that they are significantly or entirely prevented from
diffusing freely or moving with respect to one another. The reagents can
for example be placed in contact with the support, and optionally
covalently or noncovalently attached or partially/completely embedded.
The terms "microparticle," "beads" "microbeads", etc., refer to particles
(optionally but not necessarily spherical in shape) having a smallest
cross-sectional length (e.g., diameter) of 50 microns or less, preferably
10 microns or less, 3 microns or less, approximately 1 micron or less,
approximately 0.5 microns or less, e.g., approximately 0.1, 0.2, 0.3, or
0.4 microns, or smaller (e.g., under 1 nanometer, about 1-10 nanometer,
about 10-100 nanometers, or about 100-500 nanometers). Microparticles
(e.g., Dynabeads from Dynal, Oslo, Norway) may be made of a variety of
inorganic or organic materials including, but not limited to, glass
(e.g., controlled pore glass), silica, zirconia, cross-linked
polystyrene, polyacrylate, polymethylmethacrylate, titanium dioxide,
latex, polystyrene, etc. Magnetization can facilitate collection and
concentration of the microparticle-attached reagents (e.g.,
polynucleotides or ligases) after amplification, and facilitates
additional steps (e.g., washes, reagent removal, etc.). In certain
embodiments of the invention a population of microparticles having
different shapes sizes and/or colors can be used. The microparticles can
optionally be encoded, e.g., with quantum dots such that each
microparticle can be individually or uniquely identified.

[0035] As used herein the term "reaction mixture" refers to the
combination of reagents or reagent solutions, which are used to carry out
a chemical analysis or a biological assay. In some embodiments, the
reaction mixture comprises all necessary components to carry out a
nucleic acid (DNA) synthesis/amplification reaction. As described above,
such reaction mixtures may include at least one amplification primer pair
suitable for amplifying a nucleic acid sequence of interest (e.g., target
nucleic acid). As described above, a suitable reaction mixture may also
include a "master mix" containing the components (e.g., typically not
including the primer pair) needed to perform an amplification reaction
(e.g., detergent, magnesium, buffer components, etc.). Other embodiments
of reaction mixtures are also contemplated herein as would be understood
by one of skill in the art.

[0036] As used herein, the terms "reagent solution" or "solution suitable
for performing a DNA synthesis reaction" refer to any or all solutions,
which are typically used to perform an amplification reaction or DNA
synthesis. They include, but are not limited to, solutions used in DNA
amplification methods, solutions used in PCR amplification reactions, or
the like. The solution suitable for DNA synthesis reaction may comprise
buffer, salts, and/or nucleotides. It may further comprise primers and/or
DNA templates to be amplified. One or more reagent solutions are
typically included in the reactions mixtures or master mixes described
herein.

[0037] As used herein, the term "primer" or "primer sequence" refers to a
short linear oligonucleotide that hybridizes to a target nucleic acid
sequence (e.g., a DNA template to be amplified) to prime a nucleic acid
synthesis reaction. The primer may be a RNA oligonucleotide, a DNA
oligonucleotide, or a chimeric sequence (e.g., comprising RNA and DNA).
The primer may contain natural, synthetic, or modified nucleotides. Both
the upper and lower limits of the length of the primer are empirically
determined. The lower limit on primer length is the minimum length that
is required to form a stable duplex upon hybridization with the target
nucleic acid under nucleic acid amplification reaction conditions. Very
short primers (usually less than 3 nucleotides long) do not form
thermodynamically stable duplexes with target nucleic acid under such
hybridization conditions. The upper limit is often determined by the
possibility of having a duplex formation in a region other than the
pre-determined nucleic acid sequence in the target nucleic acid.
Generally, suitable primer lengths are in the range of about any of, for
example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, (and so on) nucleotides in length.

[0038] In some embodiments, the terms "probe(s)", "oligonucleotide(s)"
and/or "primer(s)" may be interchangeable terms herein, so that any one
of these may be taken as a reference to another. The terms
"polynucleotide," "oligonucleotide", "probe", "primer", "template",
"nucleic acid" and the like may be taken to refer to a populations or
pools of individual molecules that are substantially identical across
their entire length or across a relevant portion of interest. For
example, the term "template" may indicate a plurality of template
molecules that are substantially identical, etc. In the case of
polynucleotides that are degenerate at one or more positions, it will be
appreciated that the degenerate polynucleotide may comprise a plurality
of polynucleotide molecules, which have sequences that are substantially
identical only at the nondegenerate position(s) and differ in sequence at
the degenerate positions. Thus, reference to "a" polynucleotide (e.g.,
"a" primer, probe, oligonucleotide, template, etc.) may be taken to mean
a population of substantially identical polynucleotide molecules, such
that the plural nature of a population of substantially identical nucleic
acid molecules need not be explicitly indicated, but may if so desired.
These terms are also intended to provide adequate support for a claim
that explicitly specifies a single polynucleotide molecule itself.

[0039] "Ligation" involves the formation of a covalent bond or linkage
between the termini of two or more nucleic acids, e.g. oligonucleotides
and/or polynucleotides, optionally in a template-driven reaction.
Exemplary ligations may be carried out enzymatically to form a
phosphodiester linkage between a 5' carbon of a terminal nucleotide of
one oligonucleotide with 3' carbon of another oligonucleotide (e.g.,
using a ligase). The nature of the bond or linkage may vary widely and
the ligation is preferably achieved enzymatically. The efficiency of
ligation refers to the rate of ligation. Where the relative efficiency of
ligation is specified in comparative or relative terms by comparison to a
reference ligation assay, it is implicit that all other reagents and
conditions (e.g., temperature, concentration of all reagents, pH,
concentration of requisite ions such as Mg++ and Mn++, concentration of
requisite cofactors such as NAD and/or ATP, salts, buffers, molar
concentrations of all reagents, including enzyme, template, probe primer,
oligonucleotides, etc) are otherwise kept identical. For example, a
proviso that a ligase (e.g., an SFL (see below)) can ligate a short
(e.g., less than 6 nucleotides) probe at least X % (e.g., where X is 100
or less; 100, 99, 95, 90, 85, 80, 75, 70, 50, 25, 10 1, 0.5, 0.1, etc.,
or any increment in between) as efficiently as the ligase can ligate a
corresponding octanucleotide, may be understood to mean that the rate of
ligation of the shorter probe occurs at a rate that is at least X % of
the rate of ligation of the octanucleotide, where all reagents except for
the probes (e.g., primer, template, enzymes and any other reagents) and
all reaction conditions (e.g., temperature, reagent concentrations,
concentrations of any other reagents, etc) are kept invariant for
practical purposes. It is understood that ligation efficiency in absolute
or relative terms may increase or decrease depending on the exact
reaction conditions used.

[0040] Optionally, ligation is performed under in-vitro conditions that
have been experimentally determined to be suitable or optimal for ligase
activity. Preferably, reaction conditions are kept substantially similar
to in-vivo or physiological conditions in which a naturally-occurring
form of the ligase being used is naturally active. Most preferably, the
reaction conditions for a particular ligase are matched as closely as
possible to exemplary in vitro ligation conditions described herein for
that ligase. In other embodiments, the conditions are such that the
reference ligation assay produces significant or detectable ligation
within 30 minutes, within 10 minutes, within 1 minute, or within ten
seconds. Another non-limiting example of a significant or detectable rate
of ligation generates in the range of 100 pM of ligation product,
optionally about 1000 pM or 10,000 pM, in an appropriate amount of time
(e.g., 10 minutes.)

[0041] Along similar lines, it should be understood that a statement that
a result has occurred (e.g., ligation, binding) is intended to indicate
that the result has occurred at a significant or substantial level or an
enhanced level compared to when it has not occurred. For example,
ligation is said to have not occurred if it is not significant,
insubstantial or greatly reduced (e.g., reduced by at least 80%, 90%, 95%
or 99% compared to when ligation does occur (e.g., under the conditions
described in the last paragraph). In reference to ligation of two
polynucleotides, the "proximal" terminus of either polynucleotide is
other terminus that is intended to be ligated to the other
polynucleotide. It is generally the terminus that is closer to the other
polynucleotide, or the terminus that is contacted by the active site of
the ligase, or other terminus that is eventually ligated to the other
polynucleotide, while the opposite terminus is the "distal" terminus. The
terminal nucleotide residue at the proximal terminus can be termed the
proximal nucleotide, and the proximal nucleotide position optionally
designated as position 1, the penultimate nucleotide position as position
2, etc. In some non-limiting instances of template-dependent ligation,
the proximal termini of both polynucleotides are hybridized adjacently to
each other.

[0042] An exemplary type of enzymatic ligation (double-stranded ligation)
includes the formation of a covalent bond between nucleotides of a
polynucleotide (e.g., resulting in circularization) or between two or
more polynucleotides (e.g., a first double-stranded terminus of a first
polynucleotide and a second different double-stranded terminus of a
second polynucleotide). The polynucleotides may be different, or may be
the same. Polynucleotides may also be ligated using a "splint"
oligonucleotide which may be used to link nucleotides that the user
desires to ligate (e.g., on the same or different polynucleotides).
Optionally, the ends of both double-stranded termini may be joined
irrespective of their sequences (e.g., blunt-end ligation, or
non-homologous end joining).

[0043] In another variation, two double-stranded polynucleotides with
protruding single-stranded ends that are complementary to each other can
be ligated (e.g., cohesive-end ligation). In other instances, the
ligation can ligate two single-stranded polynucleotides, either or both
of which has optionally hybridized (annealed) to another nucleotide
sequence. In template-dependent ligation, ligation between a first
polynucleotide and a second polynucleotide occurs upon hybridization of
at least a portion of either or both polynucleotides to a target
sequence. The target sequence can be a portion of either polynucleotide
(e.g., self-hybridization or hybridization to each other) or to a
sequence on a third different polynucleotide (e.g., a "splint"
oligonucleotide). The hybridized portion of the polynucleotide may be,
for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20
nucleotides long. The hybridized portion is optionally a terminal portion
of the nucleotide (e.g., includes the 5' or 3' nucleotide). For example,
the hybridized portion can consist of the 5' or 3' terminal nucleotide,
or the terminal 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20 nucleotides of the 5'
or 3' end. Optionally, ligation occurs when no mismatch is present within
the hybridized portions.

[0044] In other cases, ligation occurs when one, two or three mismatches
can be present within the hybridized portion. In some cases ligation does
not occur when the terminal nucleotide and/or second-most terminal
nucleotide and/or third-most terminal nucleotide is mismatched. As
mentioned, the terminal nucleotides can be the 5'- or 3'-terminal
nucleotides of the polynucleotide. An exemplary type of assay makes use
of template-dependent ligation between a first single-stranded
polynucleotide and a second single-stranded polynucleotide, where
ligation can be effected when either or both polynucleotides is/are
hybridized to a third different single-stranded polynucleotide. In some
instances, both probes must hybridize to the template for significant
ligation to occur. For ease of reference, the first polynucleotide is
called the "initializing probe," the second polynucleotide called the
"extension probe" and the third polynucleotide called the "template."

[0045] In some variations, (e.g., "nick ligation"), both probes must
hybridize adjacently to each other on the template for ligation to occur.
In some assays, the probes are adjacently hybridized and can be ligated
only when a terminal nucleotide of the initializing probe is hybridized
to a first nucleotide of the template and a terminal nucleotide of the
extension probe is hybridized to a second nucleotide of the template,
where the first and second nucleotides on the template are not separated
by an intervening nucleotide of the template. In other embodiments, a few
intervening nucleotides may be present between the first and second
nucleotides on the template (e.g., 1, 2, 3 or more nucleotides). In such
embodiments, a "gap-filling" step can be performed to extend the 3'
terminus of one probe before it can be ligated to the 5' terminus of the
other probe.

[0046] In the methods described herein, the terminal nucleotide of the
initializing probe can be the 5' terminal nucleotide and the terminal
nucleotide of the extension probe can be the 3' terminal nucleotide.
Alternatively, the terminal nucleotide of the initializing probe can be
the 3' terminal nucleotide and the terminal nucleotide of the extension
probe can be the 5' terminal nucleotide. The ligation product of any one
reaction can optionally be subjected to further ligation and/or
non-ligation reactions in turn. For example, the ligation product can be
used as the initializing probe or extension probe or template in a
subsequent ligation. Also for example, it can be used as a template or
primer for polymerase extension, such as in polymerase chain reaction
(PCR). It can be cleaved enzymatically or chemically (for example when it
has scissile linkages), treated with exo- or endonucleases, kinases,
phosphatases, etc. The ends of a double-stranded product can be
blunt-ended or filled in, capped, or adenylated, etc.

[0047] As used herein, "splint oligonucleotide," "splint oligo," or
"connector" refers to an oligonucleotide that is used to provide an
annealing site or a "ligation template" for joining two ends of a nucleic
acid molecule or molecules using a ligase or another enzyme with ligase
activity. The ligation splint holds the ends adjacent to each other and
"creates a ligation junction" between the 5'-phosphorylated and a
3'-hydroxylated ends that are to be ligated. For example, when a ligation
splint oligo is used to join the 3'-end of a first probe oligo (oligo A)
to the 5'-end of a second probe oligo, the ligation splint oligo has a
sequence complementary to the 3'-end of oligo A (e.g., oligo tail
sequence, and a second neighboring sequence (e.g., an adjacent sequence)
that is complementary to the 5'-end of oligo B (FIG. 4).

[0048] In some embodiments of the improved PLA processes splint oligos can
be either symmetrical or asymmetrical depending on the number of
nucleotides that hybridize to each of the two oligo probes it is
connecting or ligating. FIG. 4 diagrams asymmetrical and symmetrical
splint types for use in the improved PLA processes as described herein.
In some embodiments, asymmetrical splints (or "connectors") span across
the two separate oligo probes (e.g., probe oligo A and B) with one of the
ends of the splint (e.g., either the 3'-end or the 5'-end) having more
nucleotides that hybridize to one of the probe oligos than the other end
of the splint has nucleotides that hybridize to the alternative probe
oligo (FIG. 4A). In other embodiments, symmetrical splints span across
the two separate oligo probes (e.g., probe oligo A and B) with both ends
of the splint (e.g., the 3' end and the 5' end) having equal number of
nucleotides that hybridize to each of the two probe oligos (FIG. 4BA).

[0049] Both asymmetrical and symmetrical splints can have any number of
intervening nucleotides between each of it's 3' and 5' ends that
hybridize to the separate probe oligos. Alternatively, there may be no
intervening nucleotides between each of the 3' and 5' ends that hybridize
to the probe oligos. In preferred embodiments, splint oligonucleotides
(oligos) are at least 6 nucleotides long (e.g., 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20 or more). In certain other embodiments,
each of the 3'- or 5'-ends of the splint oligo will comprise at least 3
(e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotides that separately
hybridize to (or "overlap") an oligo probe, herein referred to as the
"overhang" region.

[0050] In some embodiments of the improved PLA processes the splint oligo
is blocked at it's 3'-end. The blocking agent can be any covalently
connected moiety that prevents polymerase activity. This 3' blocked
splint oligo is then prevented from interfering with the PCR reaction
part of the improved PLA process. In some embodiments, for example, the
3' blocking agent can include, but is not limited to, 3'-fluoro-,
3'-bromo-, 3'-iodo-, 3'-deoxy-, 3'-methyl-, 3'-methoxy, 3'-phosphate,
3'-aminolink, 3'-abasic amidite, or any other 3' modification groups.
Those of ordinary skill in the art would be able to further contemplate
other blocking agents for use as disclosed herein.

[0052] A particularly useful assay is the oligonucleotide ligation assay
(OLA). The OLA is a convenient, highly-stringent method that permits
distinction among known DNA sequence variants (Landegren, 1988). For
instance, multiplex analysis of highly polymorphic loci is useful for
identification of individuals, e.g., for paternity testing and in
forensic science, organ transplant donor-receiver matching, genetic
disease diagnosis, prognosis, and pre-natal counseling, and other
genetic-based testing which depend on the discrimination of single-base
differences at a multiplicity of loci (Delahunty, 1996). Products of a
multiplex OLA may be resolved electrophoretically from one another and
from unligated probes under denaturing conditions with fluorescence
detection (Grossman, 1994). For example, two PNA-DNA chimeras, a
wild-type (WT) sequence chimera and a mutant sequence chimera, may bear
different fluorescent dyes. Only when the mutant sequence is present in
the target sample, will the mutant sequence chimera ligate to the
adjacently annealed second probe (oligo) if the mutant base pair is at
the ligation site. The ligation products may be discriminated by
separation based on: (i) size using electrophoresis or chromatography
and/or (ii) detectable labels (Grossman, 1994). With a plurality of
fluorescent dyes labeled to chimeras with sequences targeting unique
target sequences, multiplexed OLA can be conducted on a single sample in
a single vessel. Requirements for efficient multiplex OLA include probes
that anneal and ligate in a highly specific and rapid manner. The
chimeras and second probe sequences may be selected such that the mutant
base, or single base polymorphism, may be at the 5'-phosphate of the
second probe or the 3'-terminus of the chimera. It is contemplated that
OLA experiments of the present invention may be conducted on solid
supports where the template nucleic acid, PNA-DNA chimeric probe, or the
second probe may be immobilized on a solid particle or bead, or a solid
porous or non-porous surface. When immobilized, the template, chimera or
second probe is preferably covalently attached to the solid substrate,
e.g. via a terminal monomer unit. The solid substrate may be polystyrene,
controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic
beads, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene,
polyethyleneoxy, and copolymers and grafts of any of the above solid
substrates. The configuration or format of the solid substrate may be
small particles or beads of approximately 1 to 50 μm in diameter,
membranes, frits, slides, plates, micromachined chips, alkanethiol-gold
layers, non-porous surfaces, and polynucleotide-immobilizing media.

[0054] In some embodiments, the ligase is a "small footprint ligase"
(SFL). A SFL has the ability to ligate short polynucleotides (e.g., at
least about 3 nucleotides). As described herein, a SFL may ligate
oligonucleotides having a connector oligo length of as short as 3 base of
hybridized DNA adjacent to 5'-phosphate hybridized DNA. In some
embodiments, the SFL may be used to ligate oligonucleotides comprising
short overlap sequences (e.g., short connector oligo length). For
instance, the SFL may be used to ligate oligonucleotides of various
nucleotides in length, whereby each oligo has at least a 3 nucleotide
overlap with the splint oligo. Typical ligases would be better suited for
ligating longer oligonucleotides (e.g., comprising 9 or more nucleotides;
comprising 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, etc nucleotides). In
this way, oligonucleotide concentration may also be reduced to minimize
chance of solution hybridization promoted non-antigen-binding ligation.
For combining the ligation and PCR reaction into one step, ATP (cofactor
for the ligase) can be omitted from the reaction mixture. In order to
maintain the ligase function, the SFL may be pre-enriched with ATP prior
to its purification and use.

[0055] A SFL may be a naturally occurring or non-naturally occurring
(e.g., artificial, synthetic) ligase. A SFL can comprise a polypeptide
sequence that is homologous to or a variant of a known ligase sequence or
any portion thereof. Exemplary SFLs can have amino acid sequence identity
of at least 70%, optionally at least 85%, optionally at least 90 or 95%,
with a known ligase, and possesses one or more functional activities of a
ligase. A SFL can thus comprise a polypeptide having any one or more of
the following activities: (1) nucleophilic attack on ATP or NAD resulting
in release of PPi or NMN and formation of a covalent ligase-adenylate
intermediate; (2) transferring the adenylate to the 5'-end of the
5'-phosphate-terminated DNA strand to form DNA-adenylate (e.g., the
5'-phosphate oxygen of the DNA strand attacks the phosphorus of
ligase-adenylate); and, (3) formation of a covalent bond joining the
polynucleotide termini and liberation of AMP (e.g., by the attack by the
3'-OH on DNA-adenylate). Optionally, the SFL can mediate any one or more
of the following bond transformations: from phosphoanhydride (ATP) to
phosphoramidate (ligase-adenylate); from phosphoramidate
(ligase-adenylate) to phosphoanhydride (DNA-adenylate); and/or from
phosphoanhydride (DNA-adenylate) to phosphodiester (sealed DNA). The SFL
in one aspect is an enzyme that can mediate the formation of a covalent
bond between two polynucleotide termini, e.g., a 3'-OH terminus and a
5'-PO4 terminus are joined together to form a phosphodiester bond.
In some instances, DNA ligation entails any one or more of three
sequential nucleotidyl transfer steps, discussed below. All three
chemical steps depend on a divalent cation cofactor. In one aspect, the
SFL is an ATP-dependent ligase or a NAD+-dependent ligase.

[0058] As used herein, the terms "amplification", "nucleic acid
amplification", or "amplifying" refer to the production of multiple
copies of a nucleic acid template, or the production of multiple nucleic
acid sequence copies that are complementary to the nucleic acid template.
The amplification reaction may be a polymerase-mediated extension
reaction such as, for example, a polymerase chain reaction (PCR).
However, any of the known amplification reactions may be suitable for use
as described herein. The term "amplifying" that typically refers to an
"exponential" increase in target nucleic acid may be used herein to
describe both linear and exponential increases in the numbers of a select
target sequence of nucleic acid. The term "amplification reaction
mixture" and/or "master mix" may refer to an aqueous solution comprising
the various (some or all) reagents used to amplify a target nucleic acid.
Such reactions may also be performed using solid supports (e.g., an
array). The reactions may also be performed in single or multiplex format
as desired by the user. These reactions typically include enzymes,
aqueous buffers, salts, amplification primers, target nucleic acid, and
nucleoside triphosphates. Depending upon the context, the mixture can be
either a complete or incomplete amplification reaction mixture. The
method used to amplify the target nucleic acid may be any available to
one of skill in the art. Any in vitro means for multiplying the copies of
a target sequence of nucleic acid may be utilized. These include linear,
logarithmic, and/or any other amplification method. While this disclosure
may generally discuss PCR as the nucleic acid amplification reaction, it
is expected that other types of nucleic acid amplification reactions,
including both polymerase-mediated amplification reactions (such as HDA,
RPA, and RCA), as well as ligase-mediated amplification reactions (such
as LDR, LCR, and gap-versions of each), and combinations of nucleic acid
amplification reactions such as LDR and PCR (see for example U.S. Pat.
No. 6,797,470) may also be suitable. For example, in addition to those
described elsewhere herein, various ligation-mediated reactions, where
for example ligation probes are employed as opposed to PCR primers.
Additional exemplary methods include polymerase chain reaction (PCR; see,
e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and/or 5,035,996),
isothermal procedures (using one or more RNA polymerases (see, e.g., WO
2006/081222), strand displacement (see, e.g., U.S. Pat. No. RE39,007E),
partial destruction of primer molecules (see, e.g., WO2006087574)),
ligase chain reaction (LCR) (see, e.g., Wu, et al., Genomics 4: 560-569
(1990)), and/or Barany, et al. PNAS USA 88:189-193 (1991)), Qβ RNA
replicase systems (see, e.g., WO/1994/016108), RNA transcription-based
systems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g.,
U.S. Pat. No. 5,854,033; U.S. Pub. No. 2004/265897; Lizardi et al. Nat.
Genet. 19: 225-232 (1998); and/or Bailer et al. Nucleic Acid Res., 26:
5073-5078 (1998)), and strand displacement amplification (SDA) (Little,
et al. Clin Chem 45:777-784 (1999)), among others. These systems, along
with the many other systems available to the skilled artisan, may be
suitable for use in amplifying target nucleic acids for use as described
herein.

[0059] "Amplification efficiency" may refer to any product that may be
quantified to determine copy number (e.g., the term may refer to a PCR
amplicon, an LCR ligation product, and/or similar product). Reactions may
be compared by carrying out at least two separate amplification
reactions, each reaction being carried out in the absence and presence,
respectively, of a reagent and/or step and quantifying amplification that
occurs in each reaction.

[0060] Also provided are methods for amplifying a nucleic acid using at
least one polymerase, at least one primer, dNTPs, and ligating and
amplifying the target nucleic acid. In some embodiments of such methods,
at least one primer is utilized. In certain embodiments, a nucleic acid
amplification reaction mixture(s) comprising at least one polymerase,
dNTPs, and at least one primer is provided. In other embodiments, methods
for using such mixture(s) are provided. Target nucleic acids may be
amplified using any of a variety of reactions and systems. Exemplary
methods for amplifying nucleic acids include, for example,
polymerase-mediated extension reactions. For instance, the
polymerase-mediated extension reaction can be the polymerase chain
reaction (PCR). In other embodiments, the nucleic acid amplification
reaction is a multiplex reaction. For instance, exemplary methods for
amplifying and detecting nucleic acids suitable for use as described
herein are commercially available as TaqMan® (see, e.g., U.S. Pat.
Nos. 4,889,818; 5,079,352; 5,210,015; 5,436,134; 5,487,972; 5,658,751;
5,210,015; 5,487,972; 5,538,848; 5,618,711; 5,677,152; 5,723,591;
5,773,258; 5,789,224; 5,801,155; 5,804,375; 5,876,930; 5,994,056;
6,030,787; 6,084,102; 6,127,155; 6,171,785; 6,214,979; 6,258,569;
6,814,934; 6,821,727; 7,141,377; and/or 7,445,900, all of which are
hereby incorporated herein by reference in their entirety). TaqMan®
assays are typically carried out by performing nucleic acid amplification
on a target polynucleotide using a nucleic acid polymerase having 5'-3'
nuclease activity, a primer capable of hybridizing to said target
polynucleotide, and an oligonucleotide probe capable of hybridizing to
said target polynucleotide 3' relative to said primer. In some
embodiments, the oligonucleotide probe includes a detectable label (e.g.,
a fluorescent reporter molecule) and a quencher molecule capable of
quenching the fluorescence of said reporter molecule. In certain
embodiments, the detectable label and quencher molecule are part of a
single probe. As amplification proceeds, the polymerase digests the probe
to separate the detectable label from the quencher molecule. The
detectable label (e.g., fluorescence) can be monitored during the
reaction, where detection of the label corresponds to the occurrence of
nucleic acid amplification (e.g., the higher the signal the greater the
amount of amplification). Variations of TaqMan® assays (e.g., LNA®
spiked TaqMan® assay) are known in the art and would be suitable for
use in the methods described herein.

[0061] Another exemplary system suitable for use as described herein
utilizes double-stranded probes in displacement hybridization methods
(see, e.g., Morrison et al. Anal. Biochem., 18:231-244 (1989); and/or Li,
et al. Nucleic Acids Res., 30(2,e5) (2002)). In such methods, the probe
typically includes two complementary oligonucleotides of different
lengths where one includes a detectable label and the other includes a
quencher molecule. When not bound to a target nucleic acid, the quencher
suppresses the signal from the detectable label. The probe becomes
detectable upon displacement hybridization with a target nucleic acid.
Multiple probes may be used, each containing different detectable labels,
such that multiple target nucleic acids may be queried in a single
reaction.

[0062] Additional exemplary methods for amplifying and detecting target
nucleic acids suitable for use as described herein involve "molecular
beacons", which are single-stranded hairpin shaped oligonucleotide
probes. In the presence of the target sequence, the probe unfolds, binds
and emits a signal (e.g., fluoresces). A molecular beacon typically
includes at least four components: 1) the "loop", an 18-30 nucleotide
region which is complementary to the target sequence; 2) two 5-7
nucleotide "stems" found on either end of the loop and being
complementary to one another; 3) at the 5' end, a detectable label; and
4) at the 3' end, a quencher dye that prevents the detectable label from
emitting a single when the probe is in the closed loop shape (e.g., not
bound to a target nucleic acid). Thus, in the presence of a complementary
target, the "stem" portion of the beacon separates out resulting in the
probe hybridizing to the target. Other types of molecular beacons are
also known and may be suitable for use in the methods described herein.
Molecular beacons may be used in a variety of assay systems. One such
system is nucleic acid sequence-based amplification (NASBA®), a
single step isothermal process for amplifying RNA to double stranded DNA
without temperature cycling. A NASBA reaction typically requires avian
myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA
polymerase, RNase H, and two oligonucleotide primers. After
amplification, the amplified target nucleic acid may be detected using a
molecular beacon. Other uses for molecular beacons are known in the art
and would be suitable for use in the methods described herein.

[0063] The Scorpion system is another exemplary assay format that may be
used in the methods described herein. Scorpion primers are bi-functional
molecules in which a primer is covalently linked to the probe, along with
a detectable label (e.g., a fluorophore) and a quencher. In the presence
of a target nucleic acid, the detectable label and the quencher separate
which leads to an increase in signal emitted from the detectable label.
Typically, a primer used in the amplification reaction includes a probe
element at the 5' end along with a "PCR blocker" element (e.g., a
hexethylene glycol (HEG) monomer (Whitcombe, et al. Nat. Biotech. 17:
804-807 (1999)) at the start of the hairpin loop. The probe typically
includes a self-complementary stem sequence with a detectable label at
one end and a quencher at the other. In the initial amplification cycles
(e.g., PCR), the primer hybridizes to the target and extension occurs due
to the action of polymerase. The Scorpion system may be used to examine
and identify point mutations using multiple probes that may be
differently tagged to distinguish between the probes. Using PCR as an
example, after one extension cycle is complete, the newly synthesized
target region will be attached to the same strand as the probe. Following
the second cycle of denaturation and annealing, the probe and the target
hybridize. The hairpin sequence then hybridizes to a part of the newly
produced PCR product. This results in the separation of the detectable
label from the quencher and causes emission of the signal. Other uses for
molecular beacons are known in the art and would be suitable for use in
the methods described herein.

[0064] The nucleic acid polymerases that may be employed in the disclosed
nucleic acid amplification reactions may be any that function to carry
out the desired reaction including, for example, a prokaryotic, fungal,
viral, bacteriophage, plant, and/or eukaryotic nucleic acid polymerase.
As used herein, the term "DNA polymerase" refers to an enzyme that
synthesizes a DNA strand de novo using a nucleic acid strand as a
template. DNA polymerase uses an existing DNA or RNA as the template for
DNA synthesis and catalyzes the polymerization of deoxyribonucleotides
alongside the template strand, which it reads. The newly synthesized DNA
strand is complementary to the template strand. DNA polymerase can add
free nucleotides only to the 3'-hydroxyl end of the newly forming strand.
It synthesizes oligonucleotides via transfer of a nucleoside
monophosphate from a deoxyribonucleoside triphosphate (dNTP) to the
3'-hydroxyl group of a growing oligonucleotide chain. This results in
elongation of the new strand in a 5' to 3' direction. Since DNA
polymerase can only add a nucleotide onto a pre-existing 3'-OH group, to
begin a DNA synthesis reaction, the DNA polymerase needs a primer to
which it can add the first nucleotide. Suitable primers may comprise
oligonucleotides of RNA or DNA, or chimeras thereof (e.g., RNA/DNA
chimerical primers). The DNA polymerases may be a naturally occurring DNA
polymerases or a variant of natural enzyme having the above-mentioned
activity. For example, it may include a DNA polymerase having a strand
displacement activity, a DNA polymerase lacking 5' to 3' exonuclease
activity, a DNA polymerase having a reverse transcriptase activity, or a
DNA polymerase having an endonuclease activity.

[0066] In another aspect, the present disclosure provides reaction
mixtures for amplifying a nucleic acid sequence of interest (e.g., a
target sequence). In some embodiments, the reaction mixture may further
comprise a signal-generating compound (SGC) and/or detectable label. The
methods may also include one or more steps for detecting the SGC and/or
detectable label to quantitate the amplified nucleic acid.

[0067] A SGC may be a substance that is itself detectable in an assay of
choice, or capable of reacting to form a chemical or physical entity
(e.g., a reaction product) that is detectable in an assay of choice.
Representative examples of reaction products include precipitates,
fluorescent signals, compounds having a color, and the like.
Representative SGC include e.g., bioluminescent compounds (e.g.,
luciferase), fluorophores (e.g., below), bioluminescent and
chemiluminescent compounds, radioisotopes (e.g., 131I, 125I,
14C, 3H, 35S, 32P and the like), enzymes (e.g.,
below), binding proteins (e.g., biotin, avidin, streptavidin and the
like), magnetic particles, chemically reactive compounds (e.g., colored
stains), labeled oligonucleotides; molecular probes (e.g., CY3, Research
Organics, Inc.), and the like. Representative fluorophores include
fluorescein isothiocyanate, succinyl fluorescein, rhodamine B, lissamine,
9,10-diphenylanthracene, perylene, rubrene, pyrene and fluorescent
derivatives thereof such as isocyanate, isothiocyanate, acid chloride or
sulfonyl chloride, umbelliferone, rare earth chelates of lanthanides such
as Europium (Eu) and the like. Representative SGC's useful in a signal
generating conjugate include the enzymes in: TUB Class 1, especially
1.1.1 and 1.6 (e.g., alcohol dehydrogenase, glycerol dehydrogenase,
lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphate
dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and the like);
TUB Class 1.11.1 (e.g., catalase, peroxidase, amino acid oxidase,
galactose oxidase, glucose oxidase, ascorbate oxidase, diaphorase, urease
and the like); IUB Class 2, especially 2.7 and 2.7.1 (e.g., hexokinase
and the like); TUB Class 3, especially 3.2.1 and 3.1.3 (e.g., alpha
amylase, cellulase, β-galacturonidase, amyloglucosidase,
β-glucuronidase, alkaline phosphatase, acid phosphatase and the
like); TUB Class 4 (e.g., lyases); TUB Class 5 especially 5.3 and 5.4
(e.g., phosphoglucose isomerase, trios phosphatase isomerase,
phosphoglucose mutase and the like.) SGCs may also generate products
detectable by fluorescent and chemiluminescent wavelengths, e.g.,
sequencing dyes, luciferase, fluorescence emitting metals such as
152Eu, or others of the lanthanide series; compounds such as
luminol, isoluminol, acridinium salts, and the like; bioluminescent
compounds such as luciferin; fluorescent proteins (e.g., GFP or variants
thereof); and the like. Attaching certain SGC to agents can be
accomplished through metal chelating groups such as EDTA. The subject SGC
shares the common property of allowing detection and/or quantification of
an attached molecule. SGCs are optionally detectable using a visual or
optical method; preferably, with a method amenable to automation such as
a spectrophotometric method, a fluorescence method, a chemiluminescent
method, an electrical nanometric method involving e.g., a change in
conductance, impedance, resistance and the like and a magnetic field
method. Some SGCs are optionally detectable with the naked eye or with a
signal detection apparatus. Some SGCs are not themselves detectable but
become detectable when subject to further treatment. The SGC can be
attached in any manner (e.g., through covalent or non-covalent bonds) to
a binding agent of interest (e.g., an antibody or a PDZ polypeptide).
SGCs suitable for attachment to agents such as antibodies include
colloidal gold, fluorescent antibodies, Europium, latex particles, and
enzymes. The agents that bind to NS1 and NP can each comprise distinct
SGCs. For example, red latex particles can be conjugated to anti-NS1
antibodies and blue latex particles can be conjugated to anti-NP
antibodies. Other detectable SGCs suitable for use in a lateral flow
format include any moiety that is detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical,
chemical, or other means. For example, suitable SGCs include biotin for
staining with labeled streptavidin conjugate, fluorescent dyes (e.g.,
fluorescein, Texas red, rhodamine, green fluorescent protein, and the
like), radiolabels, enzymes (e.g., horseradish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric SGCs
such as colloidal gold or colored glass or plastic (e.g., polystyrene,
polypropylene, latex beads). Patents that described the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. See also Handbook of Fluorescent
Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene
Oreg.). Radiolabels can be detected using photographic film or
scintillation counters, fluorescent markers can be detected using a
photodetector to detect emitted light.

[0068] Similarly, the term "detectable label" may refer to any of a
variety of signaling molecules indicative of amplification. For example,
SYBR GREEN and other DNA-binding dyes are detectable labels. Such
detectable labels may comprise or may be, for example, nucleic acid
intercalating agents or non-intercalating agents. As used herein, an
intercalating agent is an agent or moiety capable of non-covalent
insertion between stacked base pairs of a double-stranded nucleic acid
molecule. A non-intercalating agent is one that does not insert into the
double-stranded nucleic acid molecule. The nucleic acid binding agent may
produce a detectable signal directly or indirectly. The signal may be
detectable directly using, for example, fluorescence and/or absorbance,
or indirectly using, for example, any moiety or ligand that is detectably
affected by proximity to double-stranded nucleic acid is suitable such as
a substituted label moiety or binding ligand attached to the nucleic acid
binding agent. It is typically necessary for the nucleic acid binding
agent to produce a detectable signal when bound to a double-stranded
nucleic acid that is distinguishable from the signal produced when that
same agent is in solution or bound to a single-stranded nucleic acid. For
example, intercalating agents such as ethidium bromide fluoresce more
intensely when intercalated into double-stranded DNA than when bound to
single-stranded DNA, RNA, or in solution (see, e.g., U.S. Pat. Nos.
5,994,056; 6,171,785; and/or 6,814,934). Similarly, actinomycin D
fluoresces red fluorescence when bound to single-stranded nucleic acids,
and green when bound to double-stranded nucleic acids. And in another
example, the photoreactive psoralen 4-aminomethyl-4-5'8-trimethylpsoralen
(AMT) has been reported to exhibit decreased absorption at long
wavelengths and fluorescence upon intercalation into double-stranded DNA
(Johnson et al. Photochem. & Photobiol., 33:785-791 (1981). For example,
U.S. Pat. No. 4,257,774 describes the direct binding of fluorescent
intercalators to DNA (e.g., ethidium salts, daunomycin, mepacrine and
acridine orange, 4'6-diamidino-α-phenylindole). Non-intercalating
agents (e.g., minor groove binders as described herein such as Hoechst
33258, distamycin, netropsin) may also be suitable for use. For example,
Hoechst 33258 (Searle, et al. Nuc. Acids Res. 18(13):3753-3762 (1990))
exhibits altered fluorescence with an increasing amount of target. Minor
groove binders are described in more detail elsewhere herein.

[0072] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues, or
any variant or functional fragment thereof. The terms apply to amino acid
polymers in which one or more amino acid residue is an artificial
chemical mimetic of a corresponding naturally occurring amino acid, as
well as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymers. The term "amino acid" includes naturally
occurring and synthetic amino acids, as well as amino acid analogs and
amino acid mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those encoded
by the genetic code, as well as those amino acids that are later
modified, e.g., hydroxyproline, γ-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have the
same basic chemical structure as a naturally occurring amino acid, e.g.,
an a carbon that is bound to a hydrogen, a carboxyl group, an amino
group, and an R group, e.g., homoserine, norleucine, methionine
sulfoxide, methionine methyl sulfonium. Such analogs have modified R
groups (e.g., norleucine) or modified peptide backbones, but retain the
same basic chemical structure as a naturally occurring amino acid. Amino
acid mimetics refers to chemical compounds that have a structure that is
different from the general chemical structure of an amino acid, but that
functions in a manner similar to a naturally occurring amino acid.

[0073] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or deletes a
single amino acid or a small percentage of amino acids in the encoded
sequence is a "conservatively modified variant" where the alteration
results in the substitution of an amino acid with a chemically similar
amino acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art. Such conservatively
modified variants are in addition to and do not exclude polymorphic
variants, interspecies homologs, and alleles of the invention. The
following eight groups each contain amino acids that are conservative
substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic
acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); [0078] 4)
Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine
(S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,
Creighton, Proteins (1984)). Variants of a given nucleotide sequence or
polypeptide sequence are optionally conservatively modified variants.
With respect to particular nucleic acid sequences, conservatively
modified variants refers to those nucleic acids which encode identical or
essentially identical amino acid sequences, or where the nucleic acid
does not encode an amino acid sequence, to essentially identical
sequences.

[0075] In certain applications, the antibodies may be contained within
hybridoma supernatant or ascites and utilized either directly as such or
following concentration using standard techniques. In other applications,
the antibodies may be further purified using, for example, salt
fractionation and ion exchange chromatography, or affinity chromatography
using Protein A, Protein G, Protein A/G, and/or Protein L ligands
covalently coupled to a solid support such as agarose beads, or
combinations of these techniques. The antibodies may be stored in any
suitable format, including as a frozen preparation (e.g., about
-20° C. or -70° C.), in lyophilized form, or under normal
refrigeration conditions (e.g., about 4° C.). When stored in
liquid form, it is preferred that a suitable buffer such as Tris-buffered
saline (TBS) or phosphate buffered saline (PBS) is utilized. Antibodies
and their derivatives may be incorporated into compositions (e.g.,
attached to oligonucleotides) described herein for use in vitro or in
vivo. Antibodies may also be modified for use by, for example,
biotinylation. Other methods for making and using antibodies available to
one of skill in the art may also be suitable for use.

[0076] The methods described herein may be useful for detecting and/or
quantifying a variety of target nucleic acids from a test sample (e.g.,
biological sample). A target nucleic acid is any nucleic acid for which
an assay system is designed to identify or detect as present (or not),
and/or quantify in a test sample. Such nucleic acids may include, for
example, those of infectious agents (e.g., virus, bacteria, parasite, and
the like), a disease process such as cancer, diabetes, or the like, or to
measure an immune response. Exemplary "test samples" include various
types of samples, such as biological samples. Exemplary biological
samples include, for instance, a bodily fluid (e.g., blood, saliva,
spinal fluid), a tissue sample, a food (e.g., meat) or beverage (e.g.,
milk) product, or the like. Other examples of biological samples may
include, whole blood, serum, plasma, urine, synovial fluid, saliva,
cerebrospinal fluid, tissue infiltrate, cervical or vaginal exudate,
pleural effusion, bronchioalveolar lavage fluid, gastric lavage fluid,
small or large bowel contents, and swab specimens from various bodily
orifices dispersed in a suitable medium. Expressed nucleic acids may
include, for example, genes for which expression (or lack thereof) is
associated with medical conditions such as infectious disease (e.g.,
bacterial, viral, fungal, protozoal infections) or cancer. The methods
described herein may also be used to detect contaminants (e.g., bacteria,
virus, fungus, and/or protozoan) in pharmaceutical, food, or beverage
products. The methods described herein may be also be used to detect rare
alleles in the presence of wild type alleles (e.g., one mutant allele in
the presence of 106-109 wild type alleles). The methods are
useful to, for example, detect minimal residual disease (e.g., rare
remaining cancer cells during remission, especially mutations in the p53
gene or other tumor suppressor genes previously identified within the
tumors), and/or measure mutation load (e.g., the frequency of specific
somatic mutations present in normal tissues, such as blood or urine).

[0077] Kits for performing the methods described herein are also provided.
The kit may comprise one or more probes (e.g., antibody conjugated to an
oligonucleotide) a pair of oligonucleotides for amplifying at least one
target nucleic acid from a sample, a biocatalyst (e.g., DNA polymerase)
and/or corresponding one or more probes labeled with a detectable label.
The kit may also include samples containing pre-defined target nucleic
acids to be used in control reactions. The kit may also optionally
include stock solutions, buffers, enzymes, detectable labels or reagents
required for detection, tubes, membranes, and the like that may be used
to complete the amplification reaction. In some embodiments, multiple
primer sets are included. Other embodiments of particular systems and
kits are also contemplated which would be understood by one of skill in
the art.

[0078] Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. The scope of the
present invention is not intended to be limited to this Description.

[0079] Unless otherwise apparent from the context, any feature can be
claimed in combination with any other, or be claimed as not present in
combination with another feature. A feature can be any piece of
information that can characterize an invention or can limit the scope of
a claim, for example any variation, step, feature, property, composition,
method, step, degree, level, component, material, substance, element,
mode, variable, aspect, measure, amount, option, embodiment, clause,
descriptive term, claim element or limitation.

[0080] The singular forms "a", "an" and "the" include plural referents
unless the context clearly dictates otherwise. Approximating language, as
used herein throughout the specification and claims, may be applied to
modify any quantitative representation that could permissibly vary
without resulting in a change in the basic function to which it is
related. Accordingly, a value modified by a term such as "about" is not
to be limited to the precise value specified. Where necessary, ranges
have been supplied, and those ranges are inclusive of all sub-ranges
there between.

[0081] In this disclosure, the use of the singular can include the plural
unless specifically stated otherwise or unless, as will be understood by
one of skill in the art in light of the present disclosure, the singular
is the only functional embodiment. Thus, for example, "a" may mean more
than one, and "one embodiment" may mean that the description applies to
multiple embodiments. The phrase "and/or" denotes a shorthand way of
indicating that the specific combination is contemplated in combination
and, separately, in the alternative.

[0082] It will be appreciated that there is an implied "about" prior to
the temperatures, concentrations, times, etc. discussed in the present
teachings, such that slight and insubstantial deviations are within the
scope of the present teachings herein. Also, the use of "comprise",
"comprises", "comprising", "contain", "contains", "containing",
"include", "includes", and "including" are not intended to be limiting.
It is to be understood that both the foregoing general description and
detailed description are exemplary and explanatory only and are not
restrictive of the invention.

[0083] Unless specifically noted in the above specification, embodiments
in the above specification that recite "comprising" various components
are also contemplated as "consisting of" or "consisting essentially of"
the recited components; embodiments in the specification that recite
"consisting of" various components are also contemplated as "comprising"
or "consisting essentially of" the recited components; and embodiments in
the specification that recite "consisting essentially of" various
components are also contemplated as "consisting of" or "comprising" the
recited components (this interchangeability does not apply to the use of
these terms in the claims).

[0084] Generally, features described herein are intended to be optional
unless explicitly indicated to be necessary in the specification.
Non-limiting examples of language indicating that a feature is regarded
as optional in the specification include terms such as "variation,"
"where," "while," "when," "optionally," "include," "preferred,"
"especial," "recommended," "advisable," "particular," "should,"
"alternative," "typical," "representative," "various," "such as," "the
like," "can," "may," "example," "embodiment," or "aspect," "in some,"
"example," "exemplary", "instance", "if" or any combination and/or
variation of such terms.

[0085] "Isolated" or "purified" generally refers to isolation of a
substance (compound, polynucleotide, protein, polypeptide, polypeptide
composition) such that the substance comprises a significant percent
(e.g., greater than 2%, greater than 5%, greater than 10%, greater than
20%, greater than 50%, or more, sometimes more than 90%, 95% or 99%) of
the sample in which it resides. In certain embodiments, a substantially
purified component comprises at least 50%, 80%-85%, or 90-95% of the
sample. Techniques for purifying polynucleotides and polypeptides of
interest are well-known in the art and include, for example, ion-exchange
chromatography, affinity chromatography and sedimentation according to
density. Generally, a substance is purified when it exists in a sample in
a higher proportion than it is naturally found.

[0086] Sequence identity (also called homology) refer to similarity in
sequence of two or more sequences (e.g., nucleotide or polypeptide
sequences). In the context of two or more homologous sequences, the
percent identity or homology of the sequences or subsequences thereof
indicates the percentage of all monomeric units (e.g., nucleotides or
amino acids) that are the same (e.g., about 70% identity, preferably 75%,
80%, 85%, 90%, 95% or 99% identity). The percent identity can be over a
specified region, when compared and aligned for maximum correspondence
over a comparison window, or designated region as measured using a BLAST
or BLAST 2.0 sequence comparison algorithms with default parameters
described below, or by manual alignment and visual inspection. Sequences
are said to be "substantially identical" when there is at least 90%
identity at the amino acid level or at the nucleotide level. This
definition also refers to the complement of a test sequence. Preferably,
the identity exists over a region that is at least about 25, 50, or 100
residues in length, or across the entire length of at least one compared
sequence. A preferred algorithm for determining percent sequence identity
and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are
described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977). Other
methods include the algorithms of Smith & Waterman, Adv. Appl. Math.
2:482 (1981), and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), etc.
Another indication that two nucleic acid sequences are substantially
identical is that the two molecules or their complements hybridize to
each other under stringent conditions.

[0087] Any indication that a feature is optional is intended provide
adequate support (e.g., under 35 U.S.C. 112 or Art. 83 and 84 of EPC) for
claims that include closed or exclusive or negative language with
reference to the optional feature. Exclusive language specifically
excludes the particular recited feature from including any additional
subject matter. For example, if it is indicated that A can be drug X,
such language is intended to provide support for a claim that explicitly
specifies that A consists of X alone, or that A does not include any
other drugs besides X. "Negative" language explicitly excludes the
optional feature itself from the scope of the claims. For example, if it
is indicated that element A can include X, such language is intended to
provide support for a claim that explicitly specifies that A does not
include X. Non-limiting examples of exclusive or negative terms include
"only," "solely," "consisting of," "consisting essentially of," "alone,"
"without", "in the absence of (e.g., other items of the same type,
structure and/or function)" "excluding," "not including", "not",
"cannot," or any combination and/or variation of such language.

[0088] Similarly, referents such as "a," "an," "said," or "the," are
intended to support both single and/or plural occurrences unless the
context indicates otherwise. For example "a dog" is intended to include
support for one dog, no more than one dog, at least one dog, a plurality
of dogs, etc. Non-limiting examples of qualifying terms that indicate
singularity include "a single", "one," "alone", "only one," "not more
than one", etc. Non-limiting examples of qualifying terms that indicate
(potential or actual) plurality include "at least one," "one or more,"
"more than one," "two or more," "a multiplicity," "a plurality," "any
combination of," "any permutation of," "any one or more of," etc. Claims
or descriptions that include "or" between one or more members of a group
are considered satisfied if one, more than one, or all of the group
members are present in, employed in, or otherwise relevant to a given
product or process unless indicated to the contrary or otherwise evident
from the context.

[0089] In the claims, any active verb (or its gerund) are intended to
indicate the corresponding actual or attempted action, even if no actual
action occurs. For example, the verb "hybridize" and gerund form
"hybridizing" and the like refer to actual hybridization or to attempted
hybridization by contacting nucleic acid sequences under conditions
suitable for hybridization, even if no actual hybridization occurs.
Similarly, "detecting" and "detection" when used in the claims refer to
actual detection or to attempted detection, even if no target is actually
detected.

[0090] Furthermore, it is to be understood that the inventions encompass
all variations, combinations, and permutations of any one or more
features described herein. Any one or more features may be explicitly
excluded from the claims even if the specific exclusion is not set forth
explicitly herein. It should also be understood that disclosure of a
reagent for use in a method is intended to be synonymous with (and
provide support for) that method involving the use of that reagent,
according either to the specific methods disclosed herein, or other
methods known in the art unless one of ordinary skill in the art would
understand otherwise. In addition, where the specification and/or claims
disclose a method, any one or more of the reagents disclosed herein may
be used in the method, unless one of ordinary skill in the art would
understand otherwise.

[0091] All publications and patents cited in this specification are herein
incorporated by reference in their entirety into this application as if
each individual publication or patent were specifically and individually
indicated to be incorporated by reference. Genbank records referenced by
GID or accession number, particularly any polypeptide sequence,
polynucleotide sequences or annotation thereof, are incorporated by
reference herein. The citation of any publication is for its disclosure
prior to the filing date and should not be construed as an admission that
the present invention is not entitled to antedate such publication by
virtue of prior invention.

[0092] Where ranges are given herein, the endpoints are included.
Furthermore, it is to be understood that unless otherwise indicated or
otherwise evident from the context and understanding of one of ordinary
skill in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower limit
of the range, unless the context clearly dictates otherwise.

[0093] Certain embodiments are further described in the following
examples. These embodiments are provided as examples only and are not
intended to limit the scope of the claims in any way.

EXAMPLES

Example 1

[0094] In an exemplary embodiment of typical proximity ligation assay
(PLA) processes (FIG. 1), the probe mix (e.g., comprising two probes, A
and B, (each probe comprising a streptavidin oligo "SAO" component and an
antibody "Ab" component) in probe dilution buffer "PDB") and test sample
(in sample dilution buffer "SDB") are combined into a binding reaction.
Following the binding reaction (e.g., at 37 C for 1 hour), the ligation
reaction mixture is added in order to carry out the ligation reaction. To
prepare the ligation reaction mixture, the ligase and ligation buffer are
diluted. Following the ligation reaction (e.g., at 37 C for 10 minutes),
the ligated product is stabilized by protease digestion; the protease is
then inactivated (e.g., using heat by incubation at 37 C for 10 minutes
followed by 95 C for 5 minutes). Usually, a portion of the ligated
product is transferred to the real-time PCR reaction mixture (comprising
PCR primers and proximity probe mix "PCR-PP"), then placed on the PCR
reaction plate in a qPCR instrument. Detection and quantification of the
ligated product can then proceed using standard techniques.

[0095] A schematic of an exemplary improved PLA process is illustrated in
FIG. 2. As shown therein, the binding reaction is the same as shown in
FIG. 1. However, in some embodiments of the improved processes disclosed
herein, as shown in FIG. 2, ligase is added to a real-time PCR mixture
(comprising PCR primers, proximity probe and splint mix "PCR-PPS") which
is then added directly to the binding reaction. In certain embodiments of
the improved PLA processes, a test sample (e.g., cell lysate) is
prepared, a binding reaction is allowed to take place and then a ligation
buffer added directly thereto. To that mixture is then added a proximity
probe mixture, and a PCR mixture. This combined reaction mixture is then
incubated for a suitable amount of time (e.g., room temperature for 20
minutes and then 96° C. for 5 min) and PCR is performed. The PCR
reaction mixture is then deposited onto the reaction plate in a qPCR
instrument and detection and quantification of the ligated product can
then proceed using standard techniques (as in typical PLA processes).

Example 2

[0096] Some exemplary embodiments of typical and improved PLA processes
are also compared in FIGS. 3A and 3B. As shown in the embodiments
illustrated therein, the typical process includes sample preparation, a
binding reaction, ligation, ligase inactivation using a protease,
protease inactivation (e.g., using heat), followed by real-time PCR. To
carry out the PCR step, a portion of the reaction mixture containing the
inactivated ligase and protease is transferred to the PCR plate, and the
"PCR mix" (e.g., containing primers, dNTPs, polymerase, and the like)
added thereto.

[0097] As shown in FIG. 3B, the improved process may eliminate the use of
a protease and dilution of the reaction mixture prior to PCR. As shown
therein, the ligase may be inactivated using heat, and the resultant
reaction mixture placed directly into the qPCR assay. Thus, some
embodiments of the improved PLA work flow uses entire binding reaction
products in the real-time PCR well. This provides a simplified work-flow
and reduced dilution of the reaction mixture. As a result, in some
preferred embodiments of the improved PLA processes, the PCR reaction
mixture contains a higher concentration of the ligated product (e.g., the
target nucleic acid).

[0098] In order to reduce non-binding probe ligation, the probe
concentration may be reduced. The splint (e.g., connector) oligo length
and concentration may also be reduced to minimize chance of solution
hybridization promoted by non-antigen-binding ligation (e.g., connector
oligonucleotides of at least 14 bases in length (e.g., 9 bases
overlapping a first oligo probe and 5 bases overlapping a second oligo
probe (9+5)) vs. connector oligonucleotides of at least 18 bases in
length (e.g., 9 bases overlapping a first oligo probe and 9 bases
overlapping a second oligo probe (9+9)). In such embodiments, a small
footprint ligase (SFL) may be used. As described herein, a SFL may ligate
oligonucleotides having a connector oligo length of as short as 3 bases
of hybridized DNA adjacent to 5'-phosphate hybridized DNA. For combining
the ligation and PCR reaction into one step, in some embodiments, ATP
(cofactor for the ligase) can be optionally omitted from the reaction
mixture. In order to maintain the ligase function, in other embodiments,
the SFL may be pre-enriched with ATP prior to its purification and use.

[0099] In some embodiments of the improved PLA processes splint oligos can
be used that are either considered to be symmetrical splints or
asymmetrical splints depending on the number of nucleotides that
hybridize to each of the two oligo probes it is connecting. FIG. 4
diagrams asymmetrical and symmetrical splint types for use in the
improved PLA processes as described herein. Asymmetrical splints (or
"connectors") span across the two separate oligo probes (e.g., probe
oligo A and B) with one of the ends of the splint (e.g., either the 3'
end or the 5' end) having more nucleotides that hybridize to one of the
probe oligos than the other end of the splint has nucleotides that
hybridize to the alternative probe oligo (FIG. 4A). Symmetrical splints
span across the two separate oligo probes (e.g., probe oligo A and B)
with both ends of the splint (e.g., the 3' end and the 5' end) having
equal number of nucleotides that hybridize to each of the two probe
oligos (FIG. 4BA).

[0100] Both asymmetrical and symmetrical splints can have any number of
intervening nucleotides between each of it's 3' and 5' ends that
hybridize to the separate probe oligos. Alternatively, there may be no
intervening nucleotides between each of the 3' and 5' ends that hybridize
to the probe oligos.

Example 3

[0101] FIG. 5 provides a comparison between results obtained using
exemplary embodiments of a typical process ("TaqMan Protein Assay Open
Kit from Life Technologies, Inc.; "PLA1") and an improved process (using
methods disclosed herein; "PLA2"). Both assays were set up to target CSTB
in NTera2 cell lysate. The binding reaction was identical for both PLA1
and PLA2 using the manufacturer's (Life Technologies, Inc.) recommend
reagents and protocol in 4 μl volume binding reactions. After the
binding reaction, PLA1 proceeded following the manufacturer's protocol
and reagents. The PLA2 reaction was combined with 16 μl of
ligation-PCR reaction mix. The ligation-PCR reaction mix consists of 10
μl the TaqMan Protein Assay Fast Master Mix (Life Technologies, Inc.),
1 ul Universal PCR Assay and the connector oligonucleotide 9+5 and the
SFL ligase, and 5 μl di-water. The ligation reaction was allowed to
proceed for 10 minutes. The ligated product was then placed in a
real-time PCR instrument (Step1Plus) and utilized according to the
manufacturer's instructions.

[0102] As described above, in some embodiments, the improved process
(PLA2) carries more target molecules into the PCR step (e.g., resulting
in more amplicons being generated). The improvement provided thereby is
shown in FIG. 5. As shown therein, the dCT of the improved process (PLA2)
is much improved as compared to the typical process (PLA1). In this
exemplary embodiment, the improved process provides at least about a one-
to three-fold dCt improvement over the typical process.

[0103] The improved process also provides improved assay sensitivity. As
shown by the exemplary embodiment in FIG. 6, the improved process
provides about a two- to ten-fold increase in sensitivity over the
typical process (FIG. 6). Sensitivity was calculated as the relative
quantification (RQ) fold change using the results from the typical
process as the calibrator. The dCt of the improved process was calculated
as fold improvement over the typical process. Since the RQ is calculated
from the dCt threshold of 2, and the fold-change is therefor indicative
of the improvement in sensitivity. The data show that the sensitivity of
the assay was improved by at least 2-fold, as determined using five
different targets (GFP, hCSTB, hICAM1, hLIN28, and hOCT3/4). The GFP data
was generated using the typical (e.g., PLA1) and improved (e.g., PLA2)
processes as described above using a cell lysate into which rGFP was
added (e.g., a "spiked-in" cell lysate) and a GFP probe used.

Example 4

[0104] In this example, two different splint lengths were tested at
varying concentrations.

[0105] PLA experiments were carried out using typical PLA conditions
("TaqMan Protein Assay Open Kit from Life Technologies, Inc.) according
to the manufacturers instructions, using a T4 ligase, except that splint
concentrations were varied within the range of 3.1 nM to 1000 nM. Splints
were also designed to have a two different splint lengths of 18 (9+9;
"99") or 16 (8+8; "88"). Cystatin B (CSTB) assay probes (from "TaqMan
Protein Expression Assay Kit (Human CSTB); Life Technologies, Inc.) were
used to detect either 1000 pM or 0 pM (no protein control; "NPC") of
recombinant CSTB protein in buffer. Ct values were plotted for each
splint concentration and delta Ct values (NPC Ct values minus CSTB Ct
values) and were plotted for each concentration used.

[0106] As shown in FIG. 7, a reduction in delta Ct was observed for the 99
splint at a low concentration of 3.1 nM as compared to higher
concentrations used. There was also a delta Ct observed for the 88 splint
at a concentration of 25 nM compared to higher concentrations.
Collectively, these data demonstrate that ligated products are reduced
when the splint length is decreased when the T4 ligase is used.

Example 5

[0107] In this example, five different splint lengths were tested using a
single concentration.

[0108] PLA experiments were carried out using similar methods as described
in Example 4, except that SF ligase instead of T4 ligase was used.
Briefly, splints were designed to have a different splint lengths of 12
(3+9), 13 (4+9), 14 (5+9 or 7+7), 17 (8+9), or 18 (9+9). The
concentration used for each of these splints was 100 nM. Raji lysate
("Protein Expression Lysate Control Kit from Life Technologies, Inc.) was
prepared at 500 cells/reaction or 0 cells/reaction ("NPC") and CSTB assay
probes (from "TaqMan Protein Expression Assay Kit (Human CSTB); Life
Technologies, Inc.) were used according to the manufacturer's
instructions. Ct values were plotted for each splint type and delta Ct
values (NPC Ct values minus 500 cell input Ct values) and were plotted
for each.

[0109] As shown in FIG. 8, increasing dCT was observed for splints of 12
nucleotides in length up to 14 nucleotides in length (including both
asymmetrical and symmetrical splint types). This demonstrates that SF
ligase is capable of ligating both asymmetric and symmetric splints of
both shorter and longer lengths.

Example 6

[0110] In this example, T4 ligase was compared to two different SF ligases
(e.g., SF and DLxD).

[0111] PLA experiments were carried out using similar methods as described
in Example 5, using the indicated ligases and splints of varying length,
as indicated. Briefly, splints were designed to have a two different
splint lengths of 14 (5+9; "95") or 18 (9+9; "99"). The concentration
used for each of these splints was 100 nM. Raji lysate ("Protein
Expression Lysate Control Kit from Life Technologies, Inc.) was prepared
at 500 cells/reaction or 0 cells/reaction ("NPC") and CSTB assay probes
(from "TaqMan Protein Expression Assay Kit (Human CSTB); Life
Technologies, Inc.) were used according to the manufacturer's
instructions. Ct values were plotted for each ligase and splint type and
delta Ct values (NPC Ct values minus 500 cell input Ct values) and were
plotted for each.

[0112] As shown in FIG. 9, the T4 ligase resulted no noticeable dCt using
the 5+9 splint. However, both SF ligases, SF and DLxD, were capable of
ligating the target DNA using shorter splint types. In this experiment,
SF used with the 5+9 splint resulted in the highest dCt.

[0113] The improved processes described herein, and exemplified throughout
the Examples above, provide faster times from process start to results
(fast), reduce hands-on time (simpler and cheaper), reduce lab
plasticware usage (cheaper and greener), and increased signals and
sensitivities. These improved processes provide simplified work flow by
combining ligation and PCR steps, reduced dilution factor from binding to
ligation step, reduced binding probe concentration to enable reduced
dilution factor, use of shorter connector oligo to control background
signal, use lower connector oligo concentration to control background
signal, use of SF ligase to enable use of shorter connector oligo length,
ATP enriched SF ligase purification scheme to omit ATP in ligation-PCR
step, and enabling use of the entire reaction volume to improve the PLA
signal and sensitivity.

[0114] While certain embodiments have been described in terms of the
preferred embodiments, it is understood that variations and modifications
will occur to those skilled in the art. Therefore, it is intended that
the appended claims cover all such equivalent variations that come within
the scope of the following claims.